Preclinical development of humanized monoclonal antibodies against CD169 as a broad antiviral therapeutic strategy.

New therapies to treat or prevent viral infections are essential, as recently observed during the COVID-19 pandemic. Here, we propose a therapeutic strategy based on monoclonal antibodies that block the specific interaction between the host receptor Siglec-1/CD169 and gangliosides embedded in the viral envelope. Antibodies are an excellent option for treating infectious diseases based on their high specificity, strong targeting affinity, and relatively low toxicity. Through a process of humanization, we optimized monoclonal antibodies to eliminate sequence liabilities and performed biophysical characterization. We demonstrated that they maintain their ability to block viral entry into myeloid cells. These molecular improvements during the discovery stage are key if we are to maximize efforts to develop new therapeutic strategies. Humanized monoclonal antibodies targeting CD169 provide new opportunities in the treatment of infections caused by ganglioside-containing enveloped viruses, which pose a constant threat to human health. In contrast with current neutralizing antibodies that bind antigens on the infectious particle, our antibodies can prevent several types of enveloped viruses interacting with host cells because they target the host CD169 protein, thus becoming a potential pan-antiviral therapy.

Impact of Obefazimod on Viral Persistence, Inflammation, and Immune Activation in People With Human Immunodeficiency Virus on Suppressive Antiretroviral Therapy.

BACKGROUND: Persistence of viral reservoirs has been observed in people with human immunodeficiency virus (HIV), despite long-term antiretroviral therapy (ART), and likely contributes to chronic immune activation and inflammation. Obefazimod is a novel drug that inhibits human immunodeficiency virus type 1 (HIV-1) replication and reduces inflammation. Here we assess whether obefazimod is safe and might impact HIV-1 persistence, chronic immune activation, and inflammation in ART-suppressed people with HIV. METHODS: We evaluated obefazimod-related adverse events, changes in cell-associated HIV-1 DNA and RNA, residual viremia, immunophenotype, and inflammation biomarkers in blood and rectal tissue. We compared 24 ART-suppressed people with HIV who received daily doses of 50 mg obefazimod for 12 weeks (n = 13) or 150 mg for 4 weeks (n = 11) and 12 HIV-negative individuals who received 50 mg for 4 weeks. RESULTS: The 50- and 150-mg doses of obefazimod were safe, although the 150-mg dose showed inferior tolerability. The 150-mg dose reduced HIV-1 DNA (P = .008, median fold change = 0.6) and residual viremia in all individuals with detectable viremia at baseline. Furthermore, obefazimod upregulated miR-124 in all participants and reduced the activation markers CD38, HLA-DR, and PD-1 and several inflammation biomarkers. CONCLUSIONS: The effect of obefazimod by reducing chronic immune activation and inflammation suggests a potential role for the drug in virus remission strategies involving other compounds that can activate immune cells, such as latency-reversing agents.

Actin-regulated Siglec-1 nanoclustering influences HIV-1 capture and virus-containing compartment formation in dendritic cells.

The immunoglobulin-like lectin receptor CD169 (Siglec-1) mediates the capture of HIV-1 by activated dendritic cells (DCs) through binding to sialylated ligands. These interactions result in a more efficient virus capture as compared to resting DCs, although the underlying mechanisms are poorly understood. Using a combination of super-resolution microscopy, single-particle tracking and biochemical perturbations we studied the nanoscale organization of Siglec-1 on activated DCs and its impact on viral capture and its trafficking to a single viral-containing compartment. We found that activation of DCs leads to Siglec-1 basal nanoclustering at specific plasma membrane regions where receptor diffusion is constrained by Rho-ROCK activation and formin-dependent actin polymerization. Using liposomes with varying ganglioside concentrations, we further demonstrate that Siglec-1 nanoclustering enhances the receptor avidity to limiting concentrations of gangliosides carrying sialic ligands. Binding to either HIV-1 particles or ganglioside-bearing liposomes lead to enhanced Siglec-1 nanoclustering and global actin rearrangements characterized by a drop in RhoA activity, facilitating the final accumulation of viral particles in a single sac-like compartment. Overall, our work provides new insights on the role of the actin machinery of activated DCs in regulating the formation of basal Siglec-1 nanoclustering, being decisive for the capture and actin-dependent trafficking of HIV-1 into the virus-containing compartment.

Identification of Plitidepsin as Potent Inhibitor of SARS-CoV-2-Induced Cytopathic Effect After a Drug Repurposing Screen.

There is an urgent need to identify therapeutics for the treatment of Coronavirus disease 2019 (COVID-19). Although different antivirals are given for the clinical management of SARS-CoV-2 infection, their efficacy is still under evaluation. Here, we have screened existing drugs approved for human use in a variety of diseases, to compare how they counteract SARS-CoV-2-induced cytopathic effect and viral replication in vitro. Among the potential 72 antivirals tested herein that were previously proposed to inhibit SARS-CoV-2 infection, only 18 % had an IC50 below 25 µM or 102 IU/ml. These included plitidepsin, novel cathepsin inhibitors, nelfinavir mesylate hydrate, interferon 2-alpha, interferon-gamma, fenofibrate, camostat along the well-known remdesivir and chloroquine derivatives. Plitidepsin was the only clinically approved drug displaying nanomolar efficacy. Four of these families, including novel cathepsin inhibitors, blocked viral entry in a cell-type specific manner. Since the most effective antivirals usually combine therapies that tackle the virus at different steps of infection, we also assessed several drug combinations. Although no particular synergy was found, inhibitory combinations did not reduce their antiviral activity. Thus, these combinations could decrease the potential emergence of resistant viruses. Antivirals prioritized herein identify novel compounds and their mode of action, while independently replicating the activity of a reduced proportion of drugs which are mostly approved for clinical use. Combinations of these drugs should be tested in animal models to inform the design of fast track clinical trials.

Dissemination of Mycobacterium tuberculosis is associated to a SIGLEC1 null variant that limits antigen exchange via trafficking extracellular vesicles.

The identification of individuals with null alleles enables studying how the loss of gene function affects infection. We previously described a non-functional variant in SIGLEC1, which encodes the myeloid-cell receptor Siglec-1/CD169 implicated in HIV-1 cell-to-cell transmission. Here we report a significant association between the SIGLEC1 null variant and extrapulmonary dissemination of Mycobacterium tuberculosis (Mtb) in two clinical cohorts comprising 6,256 individuals. Local spread of bacteria within the lung is apparent in Mtb-infected Siglec-1 knockout mice which, despite having similar bacterial load, developed more extensive lesions compared to wild type mice. We find that Siglec-1 is necessary to induce antigen presentation through extracellular vesicle uptake. We postulate that lack of Siglec-1 delays the onset of protective immunity against Mtb by limiting antigen exchange via extracellular vesicles, allowing for an early local spread of mycobacteria that increases the risk for extrapulmonary dissemination.

Anti-Siglec-1 antibodies block Ebola viral uptake and decrease cytoplasmic viral entry.

Several Ebola viruses cause outbreaks of lethal haemorrhagic fever in humans, but developing therapies tackle only Zaire Ebola virus. Dendritic cells (DCs) are targets of this infection in vivo. Here, we found that Ebola virus entry into activated DCs requires the sialic acid-binding Ig-like lectin 1 (Siglec-1/CD169), which recognizes sialylated gangliosides anchored to viral membranes. Blockage of the Siglec-1 receptor by anti-Siglec-1 monoclonal antibodies halted Ebola viral uptake and cytoplasmic entry, offering cross-protection against other ganglioside-containing viruses such as human immunodeficiency virus type 1.

Dendritic Cells From the Cervical Mucosa Capture and Transfer HIV-1 via Siglec-1.

Antigen presenting cells from the cervical mucosa are thought to amplify incoming HIV-1 and spread infection systemically without being productively infected. Yet, the molecular mechanism at the cervical mucosa underlying this viral transmission pathway remains unknown. Here we identified a subset of HLA-DR+ CD14+ CD11c+ cervical DCs at the lamina propria of the ectocervix and the endocervix that expressed the type-I interferon inducible lectin Siglec-1 (CD169), which promoted viral uptake. In the cervical biopsy of a viremic HIV-1+ patient, Siglec-1+ cells harbored HIV-1-containing compartments, demonstrating that in vivo, these cells trap viruses. Ex vivo, a type-I interferon antiviral environment enhanced viral capture and trans-infection via Siglec-1. Nonetheless, HIV-1 transfer via cervical DCs was effectively prevented with antibodies against Siglec-1. Our findings contribute to decipher how cervical DCs may boost HIV-1 replication and promote systemic viral spread from the cervical mucosa, and highlight the importance of including inhibitors against Siglec-1 in microbicidal strategies.

Identification of Siglec-1 null individuals infected with HIV-1.

Siglec-1/CD169 is a myeloid-cell surface receptor critical for HIV-1 capture and infection of bystander target cells. To dissect the role of SIGLEC1 in natura, we scan a large population genetic database and identify a loss-of-function variant (Glu88Ter) that is found in ∼1% of healthy people. Exome analysis and direct genotyping of 4,233 HIV-1-infected individuals reveals two Glu88Ter homozygous and 97 heterozygous subjects, allowing the analysis of ex vivo and in vivo consequences of SIGLEC1 loss-of-function. Cells from these individuals are functionally null or haploinsufficient for Siglec-1 activity in HIV-1 capture and trans-infection ex vivo. However, Siglec-1 protein truncation does not have a measurable impact on HIV-1 acquisition or AIDS outcomes in vivo. This result contrasts with the known in vitro functional role of Siglec-1 in HIV-1 trans-infection. Thus, it provides evidence that the classical HIV-1 infectious routes may compensate for the lack of Siglec-1 in fuelling HIV-1 dissemination within infected individuals.

Role of Human Organic Cation Transporter 1 (hOCT1) Polymorphisms in Lamivudine (3TC) Uptake and Drug-Drug Interactions.

Lamivudine (3TC), a drug used in the treatment of HIV infection, needs to cross the plasma membrane to exert its therapeutic action. Human Organic cation transporter 1 (hOCT1), encoded by the SLC22A1 gene, is the transporter responsible for its uptake into target cells. As SLC22A1 is a highly polymorphic gene, the aim of this study was to determine how SNPs in the OCT1-encoding gene affected 3TC internalization and its interaction with other co-administered drugs. HEK293 cells stably transfected with either the wild type form or the polymorphic variants of hOCT1 were used to perform kinetic and drug-drug interaction studies. Protein co-immunoprecipitation was used to assess the impact of selected polymorphic cysteines on the oligomerization of the transporter. Results showed that 3TC transport efficiency was reduced in all polymorphic variants tested (R61C, C88R, S189L, M420del, and G465R). This was not caused by lack of oligomerization in case of variants located at the transporter extracellular loop (R61C and C88R). Drug-drug interaction measurements showed that co-administered drugs [abacavir (ABC), zidovudine (AZT), emtricitabine (FTC), tenofovir diproxil fumarate (TDF), efavirenz (EFV) and raltegravir (RAL)], differently inhibited 3TC uptake depending upon the polymorphic variant analyzed. These data highlight the need for accurate analysis of drug transporter polymorphic variants of clinical relevance, because polymorphisms can impact on substrate (3TC) translocation but even more importantly they can differentially affect drug-drug interactions at the transporter level.

P-glycoprotein (ABCB1) activity decreases raltegravir disposition in primary CD4+P-gphigh cells and correlates with HIV-1 viral load.

OBJECTIVES: To evaluate the role of P-glycoprotein (P-gp) and multidrug-resistant-protein 1 (MRP1) on raltegravir intracellular drug disposition in CD4+ T cells, investigate the effect of HIV-1 infection on P-gp expression and correlate HIV-1 viraemia with P-gp activity in primary CD4+ T cell subsets. METHODS: The cellular accumulation ratio of [(3)H]raltegravir was quantified in CD4+ T cell lines overexpressing either P-gp (CEM-P-gp) or MRP1 (CEM-MRP1) and in primary CD3+CD4+ T cells with high (P-gp(high)) and low P-gp activity (P-gp(low)); inhibition of efflux transporters was confirmed by the intracellular retention of calcein-AM. The correlation of P-gp activity with HIV-1 viraemia was assessed in naive and memory T cell subsets from 21 HIV-1-infected treatment-naive subjects. RESULTS: [(3)H]Raltegravir cellular accumulation ratio decreased in CEM-P-gp cells (P < 0.0001). XR9051 (a P-gp inhibitor) and HIV-1 PIs reversed this phenomenon. Primary CD4+P-gp(high) cells accumulated less raltegravir (38.4% ±â€Š9.6%) than P-gp(low) cells, whereas XR9051 also reversed this effect. In vitro HIV-1 infection of PBMCs and stimulation of CD4+ T cells increased P-gp mRNA and P-gp activity, respectively, while primary CD4+P-gp(high) T cells sustained a higher HIV-1 replication than P-gp(low) cells. A significant correlation between HIV-1 viraemia and P-gp activity was found in different CD4+ T cell subsets, particularly memory CD4+ T cells (r = 0.792, P < 0.0001). CONCLUSIONS: Raltegravir is a substrate of P-gp in CD4+ T cells. Primary CD4+P-gp(high) T cells eliminate intracellular raltegravir more readily than P-gp(low) cells and HIV-1 viraemia correlates with P-gp overall activity. Specific CD4+P-gp(high) T cell subsets could facilitate the persistence of viral replication in vivo and ultimately promote the appearance of drug resistance.

Long-Term Spontaneous Control of HIV-1 Is Related to Low Frequency of Infected Cells and Inefficient Viral Reactivation.

UNLABELLED: HIV establishes reservoirs of infected cells that persist despite effective antiretroviral therapy (ART). In most patients, the virus begins to replicate soon after treatment interruption. However, a low frequency of infected cells at the time of treatment interruption has been associated with delayed viral rebound. Likewise, individuals who control the infection spontaneously, so-called HIV-1 controllers (HICs), carry particularly low levels of infected cells. It is unclear, however, whether and how this small number of infected cells contributes to durable viral control. Here we compared 38 HICs with 12 patients on effective combined antiretroviral therapy (cART) and found that the low frequency of infected cells in the former subjects was associated both with less efficient viral reactivation in resting CD4(+) T cells and with less efficient virion production ex vivo We also found that a potent HIV-specific CD8(+) T cell response was present only in those HICs whose CD4(+) T cells produced virus ex vivo Long-term spontaneous control of HIV infection in HICs thus appears to be sustained on the basis of the inefficient reactivation of viruses from a limited number of infected cells and the capacity of HICs to activate a potent HIV-specific CD8(+) T cell response to counteract efficient viral reactivation events. IMPORTANCE: There is a strong scientific interest in developing strategies to eradicate the HIV-1 reservoir. Very rare HIV-1-infected patients are able to spontaneously control viremia for long periods of time (HIV-1 controllers [HICs]) and are put forward as a model of HIV-1 remission. Here, we show that the low viral reservoirs found in HICs are a critical part of the mechanisms underlying viral control and result in a lower probability of HIV-1 reactivation events, resulting in limited HIV-1 release and spread. We found that those HICs in whom viral reactivation and spread from CD4(+) T cells in vitro were the most difficult were those with diminished CD8(+) T cell responses. These results suggest that, in some settings, low HIV-1 reservoirs decisively contribute to at least the temporary control of infection without antiretroviral therapy. We believe that this work provides information of relevance in the context of the search for HIV-1 remission.

HIV-1 immune activation induces Siglec-1 expression and enhances viral trans-infection in blood and tissue myeloid cells.

BACKGROUND: Myeloid cells are key players in the recognition and response of the host against invading viruses. Paradoxically, upon HIV-1 infection, myeloid cells might also promote viral pathogenesis through trans-infection, a mechanism that promotes HIV-1 transmission to target cells via viral capture and storage. The receptor Siglec-1 (CD169) potently enhances HIV-1 trans-infection and is regulated by immune activating signals present throughout the course of HIV-1 infection, such as interferon α (IFNα). RESULTS: Here we show that IFNα-activated dendritic cells, monocytes and macrophages have an enhanced ability to capture and trans-infect HIV-1 via Siglec-1 recognition of viral membrane gangliosides. Monocytes from untreated HIV-1-infected individuals trans-infect HIV-1 via Siglec-1, but this capacity diminishes after effective antiretroviral treatment. Furthermore, Siglec-1 is expressed on myeloid cells residing in lymphoid tissues, where it can mediate viral trans-infection. CONCLUSIONS: Siglec-1 on myeloid cells could fuel novel CD4(+) T-cell infections and contribute to HIV-1 dissemination in vivo.

Highly pathogenic adapted HIV-1 strains limit host immunity and dictate rapid disease progression.

OBJECTIVE: The study of HIV-1 rapid progressors has been limited to specific case reports. Nevertheless, identification and characterization of the viral and host factors involved in rapid progression are crucial when attempting to uncover the correlates of rapid disease outcome. DESIGN: We carried out comparative functional analyses in rapid progressors (n = 46) and standard progressors (n = 46) early after HIV-1 seroconversion (≤1 year). The viral traits tested were viral replicative capacity, co-receptor usage, and genomic variation. Host CD8(+) T-cell responses, humoral activity, and HLA immunogenetic markers were also determined. RESULTS: Our data demonstrate an unusual convergence of highly pathogenic HIV-1 strains in rapid progressors. Compared with standard progressors, rapid progressor viral strains show higher in-vitro replicative capacity (81.5 vs. 67.9%; P = 0.025) and greater X4/DM co-receptor usage (26.3 vs. 2.8%; P = 0.006) in early infection. Limited or absent functional HIV-1 CD8(+) T-cell responses and neutralizing activity were measured in rapid progressors. Moreover, the increase in common HLA allele-restricted CD8(+) T-cell escape mutations in rapid progressors acts as a signature of uncontrolled HIV-1 replication and early impairment of adaptive cellular responses. CONCLUSION: Our data support a dominant role for viral factors in rapid progressors. Robust HIV-1 replication and intrinsic viral properties limit host adaptive immune responses, thus driving rapid disease progression.

The infectious synapse formed between mature dendritic cells and CD4(+) T cells is independent of the presence of the HIV-1 envelope glycoprotein.

BACKGROUND: Since cell-mediated infection of human immunodeficiency virus type 1 (HIV-1) is more efficient than cell-free infection, cell-to-cell propagation plays a crucial role in the pathogenesis of HIV-1 infection. Transmission of HIV-1 is enabled by two types of cellular contacts, namely, virological synapses between productively infected cells and uninfected target cells and infectious synapses between uninfected dendritic cells (DC) harboring HIV-1 and uninfected target cells. While virological synapses are driven by expression of the viral envelope glycoprotein on the cell surface, little is known about the role of envelope glycoprotein during contact between DC and T cells. We explored the contribution of HIV-1 envelope glycoprotein, adhesion molecules, and antigen recognition in the formation of conjugates comprising mature DC (mDC) and CD4(+) T cells in order to further evaluate their role in mDC-mediated HIV-1 transmission at the immunological synapse. RESULTS: Unlike virological synapse, HIV-1 did not modulate the formation of cell conjugates comprising mDC harboring HIV-1 and non-activated primary CD4(+) T cells. Disruption of interactions between ICAM-1 and LFA-1, however, resulted in a 60% decrease in mDC-CD4(+) T-cell conjugate formation and, consequently, in a significant reduction of mDC-mediated HIV-1 transmission to non-activated primary CD4(+) T cells (p < 0.05). Antigen recognition or sustained MHC-TcR interaction did not enhance conjugate formation, but significantly boosted productive mDC-mediated transmission of HIV-1 (p < 0.05) by increasing T-cell activation and proliferation. CONCLUSIONS: Formation of the infectious synapse is independent of the presence of the HIV-1 envelope glycoprotein, although it does require an interaction between ICAM-1 and LFA-1. This interaction is the main driving force behind the formation of mDC-CD4(+) T-cell conjugates and enables transmission of HIV-1 to CD4(+) T cells. Moreover, antigen recognition boosts HIV-1 replication without affecting the frequency of cellular conjugates. Our results suggest a determinant role for immune activation driven by mDC-CD4(+) T-cell contacts in viral dissemination and that this activation likely contributes to the pathogenesis of HIV-1 infection.

Siglec-1 is a novel dendritic cell receptor that mediates HIV-1 trans-infection through recognition of viral membrane gangliosides.

Dendritic cells (DCs) are essential antigen-presenting cells for the induction of immunity against pathogens. However, HIV-1 spread is strongly enhanced in clusters of DCs and CD4(+) T cells. Uninfected DCs capture HIV-1 and mediate viral transfer to bystander CD4(+) T cells through a process termed trans-infection. Initial studies identified the C-type lectin DC-SIGN as the HIV-1 binding factor on DCs, which interacts with the viral envelope glycoproteins. Upon DC maturation, however, DC-SIGN is down-regulated, while HIV-1 capture and trans-infection is strongly enhanced via a glycoprotein-independent capture pathway that recognizes sialyllactose-containing membrane gangliosides. Here we show that the sialic acid-binding Ig-like lectin 1 (Siglec-1, CD169), which is highly expressed on mature DCs, specifically binds HIV-1 and vesicles carrying sialyllactose. Furthermore, Siglec-1 is essential for trans-infection by mature DCs. These findings identify Siglec-1 as a key factor for HIV-1 spread via infectious DC/T-cell synapses, highlighting a novel mechanism that mediates HIV-1 dissemination in activated tissues.

Sialyllactose in viral membrane gangliosides is a novel molecular recognition pattern for mature dendritic cell capture of HIV-1.

HIV-1 is internalized into mature dendritic cells (mDCs) via an as yet undefined mechanism with subsequent transfer of stored, infectious virus to CD4+ T lymphocytes. Thus, HIV-1 subverts a DC antigen capture mechanism to promote viral spread. Here, we show that gangliosides in the HIV-1 membrane are the key molecules for mDC uptake. HIV-1 virus-like particles and liposomes mimicking the HIV-1 lipid composition were shown to use a common internalization pathway and the same trafficking route within mDCs. Hence, these results demonstrate that gangliosides can act as viral attachment factors, in addition to their well known function as cellular receptors for certain viruses. Furthermore, the sialyllactose molecule present in specific gangliosides was identified as the determinant moiety for mDC HIV-1 uptake. Thus, sialyllactose represents a novel molecular recognition pattern for mDC capture, and may be crucial both for antigen presentation leading to immunity against pathogens and for succumbing to subversion by HIV-1.

HIV-1 capture and antigen presentation by dendritic cells: enhanced viral capture does not correlate with better T cell activation.

During HIV-1 infection, dendritic cells (DC) facilitate dissemination of HIV-1 while trying to trigger adaptive antiviral immune responses. We examined whether increased HIV-1 capture in DC matured with LPS results in more efficient Ag presentation to HIV-1-specific CD4(+) and CD8(+) T cells. To block the DC-mediated trans-infection of HIV-1 and maximize Ag loading, we also evaluated a noninfectious integrase-deficient HIV-1 isolate, HIV(NL4-3ΔIN). We showed that higher viral capture of DC did not guarantee better Ag presentation or T cell activation. Greater HIV(NL4-3) uptake by fully LPS-matured DC resulted in higher viral transmission to target cells but poorer stimulation of HIV-1-specific CD4(+) and CD8(+) T cells. Conversely, maturation of DC with LPS during, but not before, viral loading enhanced both HLA-I and HLA-II HIV-1-derived Ag presentation. In contrast, DC maturation with the clinical-grade mixture consisting of IL-1ß, TNF-α, IL-6, and PGE(2) during viral uptake only stimulated HIV-1-specific CD8(+) T cells. Hence, DC maturation state, activation stimulus, and time lag between DC maturation and Ag loading impact HIV-1 capture and virus Ag presentation. Our results demonstrate a dissociation between the capacity to capture HIV-1 and to present viral Ags. Integrase-deficient HIV(NL4-3ΔIN) was also efficiently captured and presented by DC through the HLA-I and HLA-II pathways but in the absence of viral dissemination. HIV(NL4-3ΔIN) seems to be an attractive candidate to be explored. These results provide new insights into DC biology and have implications in the optimization of DC-based immunotherapy against HIV-1 infection.

In-depth characterization of viral isolates from plasma and cells compared with plasma circulating quasispecies in early HIV-1 infection.

BACKGROUND: The use of in vitro models to unravel the phenotypic characteristics of circulating viral variants is key to understanding HIV-1 pathogenesis but limited by the availability of primary viral isolates from biological samples. However, overall in vivo genetic variability of HIV-1 within a subject may not be reflected in the viable viral population obtained after isolation. Although several studies have tried to determine whether viral populations expanded in vitro are representative of in vivo findings, the answer remains unclear due to the reduced number of clonal sequences analyzed or samples compared. In order to overcome previous experimental limitations, here we applied Deep Pyrosequencing (DPS) technology in combination with phenotypic experiments to analyze and compare with unprecedented detail the composition of viral isolates and in vivo quasispecies. METHODOLOGY/PRINCIPAL FINDINGS: We amplified by DPS HIV-1 genomic regions covering gag, protease, integrase and env-V3 to characterize paired isolates from plasma and peripheral blood mononuclear cells and compare them with total plasma viral RNA in four recently HIV-1 infected subjects. Our study demonstrated the presence of unique haplotypes scattered between sample types with conservation of major variants. In addition, no differences in intra- and inter-population encoded protein variability were found between the different types of isolates or when these were compared to plasma viral RNA within subjects. Additionally, in vitro experiments demonstrated phenotypic similarities in terms of replicative capacity and co-receptor usage between viral isolates and plasma viral RNA. CONCLUSION: This study is the first in-depth comparison and characterization of viral isolates from different sources and plasma circulating quasispecies using DPS in recently HIV-1 infected subjects. Our data supports the use of primary isolates regardless of their plasma or cellular origin to define genetic variability and biological traits of circulating HIV-1 quasispecies.